Bottom Line:
Fractures and defects of 10% and 20% of the total limb length regenerated well without any intervention, but 40% and 50% defects failed to regenerate after either simple removal of bone or implanting SIS scaffold alone.By contrast, scaffold soaked in the growth factor combination BMP-4/HGF or in protein extract of intact limb tissue promoted partial or extensive induction of cartilage and bone across 50% segment defects in 30%-33% of cases.These results show that BMP-4/HGF and intact tissue protein extract can promote the events required to induce cartilage and bone formation across a segment defect larger than critical size and that the long bones of axolotl limbs are an inexpensive model to screen soluble factors and natural and synthetic scaffolds for their efficacy in stimulating this process.

Affiliation: Department of Biology, School of Science, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana, United States of America.

ABSTRACTWe tested the ability of the axolotl (Ambystoma mexicanum) fibula to regenerate across segment defects of different size in the absence of intervention or after implant of a unique 8-braid pig small intestine submucosa (SIS) scaffold, with or without incorporated growth factor combinations or tissue protein extract. Fractures and defects of 10% and 20% of the total limb length regenerated well without any intervention, but 40% and 50% defects failed to regenerate after either simple removal of bone or implanting SIS scaffold alone. By contrast, scaffold soaked in the growth factor combination BMP-4/HGF or in protein extract of intact limb tissue promoted partial or extensive induction of cartilage and bone across 50% segment defects in 30%-33% of cases. These results show that BMP-4/HGF and intact tissue protein extract can promote the events required to induce cartilage and bone formation across a segment defect larger than critical size and that the long bones of axolotl limbs are an inexpensive model to screen soluble factors and natural and synthetic scaffolds for their efficacy in stimulating this process.

pone.0130819.g007: Result of implanting 8-braid SIS scaffold soaked in 0.8 aPBS.Left, 8-braid scaffold prior to hydration. Middle and right, 50% defect at one and two months, respectively, after embedding SIS scaffold alone. No cartilage has regenerated. In the one-month specimen, the implanted scaffold is still visible within the gap (arrow). At two months, the scaffold is largely degraded, and connective tissue and muscle has regenerated into the gap (arrow). M = muscle. Scale bar = 400 μm.

Mentions:
To determine whether the 8-braid SIS scaffold would by itself promote regeneration across 40% and 50% defects, we implanted four scaffolds into 40% defects and 16 scaffolds into 50% defects. The braid was hydrated by dipping it into the growth factor solvent (sterile 0.8x aPBS) prior to insertion into the defect. Trichrome and H&E staining of sections were used to monitor degradation of the scaffold and the tissue filling the defect space. After three months there was no visible skeletal regeneration in any of the defects and they were filled with disorganized connective tissue and regenerated muscle (Fig 7). These results demonstrate that the SIS scaffold was unable by itself to stimulate regeneration of skeletal tissue.

pone.0130819.g007: Result of implanting 8-braid SIS scaffold soaked in 0.8 aPBS.Left, 8-braid scaffold prior to hydration. Middle and right, 50% defect at one and two months, respectively, after embedding SIS scaffold alone. No cartilage has regenerated. In the one-month specimen, the implanted scaffold is still visible within the gap (arrow). At two months, the scaffold is largely degraded, and connective tissue and muscle has regenerated into the gap (arrow). M = muscle. Scale bar = 400 μm.

Mentions:
To determine whether the 8-braid SIS scaffold would by itself promote regeneration across 40% and 50% defects, we implanted four scaffolds into 40% defects and 16 scaffolds into 50% defects. The braid was hydrated by dipping it into the growth factor solvent (sterile 0.8x aPBS) prior to insertion into the defect. Trichrome and H&E staining of sections were used to monitor degradation of the scaffold and the tissue filling the defect space. After three months there was no visible skeletal regeneration in any of the defects and they were filled with disorganized connective tissue and regenerated muscle (Fig 7). These results demonstrate that the SIS scaffold was unable by itself to stimulate regeneration of skeletal tissue.

Bottom Line:
Fractures and defects of 10% and 20% of the total limb length regenerated well without any intervention, but 40% and 50% defects failed to regenerate after either simple removal of bone or implanting SIS scaffold alone.By contrast, scaffold soaked in the growth factor combination BMP-4/HGF or in protein extract of intact limb tissue promoted partial or extensive induction of cartilage and bone across 50% segment defects in 30%-33% of cases.These results show that BMP-4/HGF and intact tissue protein extract can promote the events required to induce cartilage and bone formation across a segment defect larger than critical size and that the long bones of axolotl limbs are an inexpensive model to screen soluble factors and natural and synthetic scaffolds for their efficacy in stimulating this process.

Affiliation:
Department of Biology, School of Science, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana, United States of America.

ABSTRACTWe tested the ability of the axolotl (Ambystoma mexicanum) fibula to regenerate across segment defects of different size in the absence of intervention or after implant of a unique 8-braid pig small intestine submucosa (SIS) scaffold, with or without incorporated growth factor combinations or tissue protein extract. Fractures and defects of 10% and 20% of the total limb length regenerated well without any intervention, but 40% and 50% defects failed to regenerate after either simple removal of bone or implanting SIS scaffold alone. By contrast, scaffold soaked in the growth factor combination BMP-4/HGF or in protein extract of intact limb tissue promoted partial or extensive induction of cartilage and bone across 50% segment defects in 30%-33% of cases. These results show that BMP-4/HGF and intact tissue protein extract can promote the events required to induce cartilage and bone formation across a segment defect larger than critical size and that the long bones of axolotl limbs are an inexpensive model to screen soluble factors and natural and synthetic scaffolds for their efficacy in stimulating this process.